PROJECT SUMMARY/ABSTRACT Despite significant advances in their surgical management, congenital cardiac anomalies remain a leading cause of death in the newborn period. Infants diagnosed with congenital heart disease often require surgical intervention in the first years of life. Currently used synthetic materials used in these operations are plagued by poor durability due to neointimal hyperplasia, experience a high incidence of thromboembolism, are susceptible to infection, and perhaps most importantly, lack growth capacity. These complications often require re-operation, which accrue the cost of increased morbidity and mortality. The development and translation of a tissue-engineered vascular graft (TEVG) with growth potential offers a solution to this pressing problem and could substantially improve outcomes in children requiring congenital heart surgery by reducing the number of graft related complications, eliminating the risk of serial re-operation, and enabling earlier, definitive surgical repair of congenital cardiac anomalies. A completely autologous TEVG has recently demonstrated the ability to grow and remodel in the pediatric population. However, TEVGs currently suffer a high incidence of stenosis, or inward narrowing, which limits their widespread clinical adoption. Our team discovered that neovessel formation after TEVG implantation is an inflammation-mediated process of vascular remodeling. Paradoxically, TEVG stenosis is caused by excessive host inflammation, suggesting that there exists a proper balance of macrophage activity that permits successful neovessel formation while simultaneously eluding the development of stenosis. The mechanism underlying this equilibrium is unknown, but recent evidence strongly implicates the LYST protein as a regulator of inflammatory macrophage function in the developing TEVG neotissue. I thus propose to investigate how LYST protein function in macrophages underlies the excessive neotissue formation observed in TEVG stenosis and validate a novel strategy to inhibit its progression via the following three specific aims: 1. Elucidate the contribution of LYST to excessive vascular inflammation causing TEVG stenosis, 2. Determine if LYST activity in monocytes and macrophages causes TEVG stenosis, and 3. Target anti-LYST therapy to infiltrating mononuclear phagocytes. Successful completion of these aims would provide a foundation for the development of a safe, effective, and rationally designed strategy to prevent TEVG stenosis and overcome the critical barrier preventing widespread adoption of tissue-engineered vascular grafts for treatment of the most severe forms of congenital heart disease.